Project Summary: This fellowship application seeks support for the research of an individual postdoctoral fellow and proposes a training plan that will equip him to become an independent scientist at a major research institution. The research project aims to elucidate the biochemical and cellular mechanisms of a recently discovered bacterial defense mechanism, called CRISPR, which has not only deepened our understanding of bacterial genetics and evolution, but also revolutionized approaches in genome editing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), along with CRISPR-associated (cas) genes, is a prokaryotic adaptive immune system that can cleave foreign genetic elements in a sequence specific manner. CRISPR-Cas systems have been classified into six distinct types (I-VI), depending on the content of their cas genes. Of the six types, the type III-A CRISPR-Cas system is unique in that it targets both single-stranded DNA and RNA, and that it requires active transcription of the target sequence. However, little is known about how the type III-A system is able to access and target its DNA substrate during the course of transcription. Aim 1 of this proposal strives to tackle this question by employing crosslinking experiments coupled with mass spectrometry to map the interaction interface between the type III-A CRISPR-Cas effector complex (Cas10-Cas) and the RNA polymerase (RNAP) elongation complex. Part one of Aim 1 focuses on how Cas10-Csm interacts with the exposed non-template DNA strand during elongation, while part two investigates whether the RNAP and Cas10-Csm complexes form any specific protein-protein interactions. These experiments will provide the first structural insights into how Cas10-Csm engages with the transcription machinery, and offer valuable training in the biochemistry and structural biology of large macromolecular complexes. Aim 2 tackles the problem of DNA targeting by the type III-A CRISPR-Cas system in living cells. In particular, major questions remain regarding how the single-stranded DNase activity of Cas10-Csm affects the genomic integrity of the bacterium and whether other cellular factors can influence the efficacy of DNA targeting. Part one of Aim 2 focuses on examining the effects of type III-A CRISPR targeting on the genomic integrity of the bacterium, employing well- established cellular methods for detecting DNA damage. Part two of Aim 2 seeks to identify genetic factors that can either promote or abrogate type III-A CRISPR DNA targeting using transposon insertion mutagenesis coupled with deep-sequencing (Tnseq). These experiments will establish how the type III-A CRISPR-Cas system functions in concert with other cellular processes, such as DNA repair and degradation, while also offering training experience in advanced molecular biology techniques. The mechanistic insights on the type III- A CRISPR-Cas system the gathered by this proposal could assist in efforts to translate this unique bacterial defense system into biotechnological tools that can improve human health.